Neonatal seizures have an incidence of 3.5 per 1000 newborns; while hypoxic-ischemic encephalopathy (HIE) accounts for 50–60% of cases, half are resistant to 1st-line anti-seizure drugs such as phenobarbital (PB). Tyrosine receptor kinase B (TrkB) activation following ischemic injury is known to increase neuronal excitability by downregulation of K-Cl co-transporter 2 (KCC2); a neuronal chloride (Cl−) co-transporter. In this study, three graded doses of ANA12, a small-molecule selective TrkB antagonist, were tested in CD1 mice at P7 and P10 following induction of neonatal ischemia by a unilateral carotid ligation. The PB loading dose remained the same in all treatment groups at both ages. Evaluation criteria for the anti-seizure efficacy of ANA12 were: (1) quantitative electroencephalographic (EEG) seizure burden and power, (2) rescue of post-ischemic KCC2 and pKCC2-S940 downregulation and (3) reversal of TrkB pathway activation following ischemia. ANA12 significantly rescued PB resistant seizures in a dose-dependent manner at P7 and improved PB efficacy at P10. Additionally, female pups responded better to lower doses of ANA12 compared to males. ANA12 significantly reversed post-ischemic KCC2 downregulation and TrkB pathway activation at P7 when PB alone was inefficacious. Rescuing KCC2 hypofunction may be critical for preventing emergence of refractory seizures.

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Neonatal seizures are harmful to the developing brain and are associated with mortality and long-term neurological comorbidities. Hypoxic-ischemic encephalopathy (HIE) seizures represent a significant proportion of such seizures. Phenobarbital (PB) remains the first line anti-seizure drug (ASD) treatment but fails ~50% of the time. Translational models of neonatal seizures are crucial to investigating mechanisms underlying PB-resistance. A model of PB-resistant ischemic seizures in postnatal day 7 (P7) CD-1 mice reported K-Cl cotransporter 2 (KCC2) degradation that has been shown to be due to activation of the TrkB pathway. We investigated PB-efficacy in a pentylenetetrazole (PTZ) model of neonatal seizures in the same strain and age using identical treatment protocols to gain insights into mechanisms underlying PB-resistance. A single dose of PTZ (80 mg/kg; IP) consistently induced repetitive seizures that did not progress to status epilepticus (SE). PB (25 mg/kg; IP, single dose) significantly suppressed the PTZ-induced seizures. This was associated with significant KCC2 upregulation and stable Na-K-Cl cotransporter 1 (NKCC1) expression at 24h. The TrkB pathway was not activated. PTZ seizure burdens were significantly higher than those reported for ischemic seizures, indicating seizure severity did not dictate the differences in PB-efficacy. Bumetanide (BTN) (0.1-0.2 mg/kg; IP) did not work as an anti-seizure agent, similar to the ischemic model. When investigating mechanisms underlying the emergence of PB-resistance in translational models, the method by which seizures are induced may dictate mechanisms underlying emergence of PB-resistance.

The drug CLP290 may treat neonatal seizures by acutely enhancing the function of K + /Cl − cotransporter KCC2.

Neonatal seizures pose a clinical challenge for their early detection, acute management, and mitigation of long-term comorbidities. A major cause of neonatal seizures is hypoxic-ischemic encephalopathy that results in seizures that are frequently refractory to the first-line anti-seizure medication phenobarbital (PB). One proposed mechanism for PB-inefficacy during neonatal seizures is the reduced expression and function of the neuron-specific K+/Cl− cotransporter 2 (KCC2), the main neuronal Cl− extruder that maintains chloride homeostasis and influences the efficacy of GABAergic inhibition. To determine if PB-refractoriness after ischemic neonatal seizures is dependent upon KCC2 hypofunction and can be rescued by KCC2 functional enhancement, we investigated the recently developed KCC2 functional enhancer CLP290 in a CD-1 mouse model of refractory ischemic neonatal seizures quantified with vEEG. We report that acute CLP290 intervention can rescue PB-resistance, KCC2 expression, and the development of epileptogenesis after ischemic neonatal seizures. KCC2 phosphorylation sites have a strong influence over KCC2 activity and seizure susceptibility in adult experimental epilepsy models. Therefore, we investigated seizure susceptibility in two different knock-in mice in which either phosphorylation of S940 or T906/T1007 was prevented. We report that KCC2 phosphorylation regulates both neonatal seizure susceptibility and CLP290-mediated KCC2 functional enhancement. Our results validate KCC2 as a clinically relevant target for refractory neonatal seizures and provide insights for future KCC2 drug development.